Blood clotting begins when platelets adhere to the cut wall of an injured blood vessel at a lesion site. Subsequently, in a cascade of enzymatically regulated reactions, soluble fibrinogen molecules are converted by the enzyme thrombin to insoluble strands of fibrin that hold the platelets together in a thrombus. At each step in the cascade, a variant precursor is converted to a protease that cleaves the next variant precursor in the series. Cofactors are required at most of the steps.
Factor VIII (also called antihemophilic factor VIII or FVIII) circulates as an inactive precursor in blood, bound tightly and non-covalently to von Willebrand factor. Factor VIII is proteolytically activated by thrombin or factor Xa, which dissociates it from von Willebrand factor and activates its procoagulant function in the cascade. In its active form, the variant factor VIIIa is a cofactor that increases the catalytic efficiency of factor IXa toward factor X activation by several orders of magnitude.
Cloning of human FVIII has revealed that the variant contains 2332 amino acids organized within a number of domains with the sequence A1-A2-B-A3-C1-C2 (Vehar et al., 1984, Nature 312, 337-342; Toole et al., 1984, Nature 312, 342-347 and Wood et al., 1984, Nature 312, 330-337). Most of FVIII is heterodimeric in plasma, containing a light-chain and various heavy-chain derivatives. The heterodimeric structure is due to proteolytic cleavage of the precursor at arginine1648, resulting in heavy- and light-chains comprising A1-A2-B and A3-C1-C2, respectively. The heterogeneity within the heavy chain is explained by limited proteolysis within its carboxy-terminal B-domain.
In order to function as a co-factor for factor X activation, FVIII requires limited proteolysis by factor Xa or thrombin. This activation involves cleavage at arginine at positions 372 and 740 on the heavy-chain and at position 1689 on the light-chain. It has been established that in comparison with the inactive precursor, active FVIII cofactor lacks a light chain fragment 1649-1689 and the whole B-domain (Mertens et al., 1993).
People with deficiencies in factor VIII or antibodies against factor VIII who are not treated with factor VIII suffer uncontrolled internal bleeding that may cause a range of serious symptoms, from inflammatory reactions in joints to early death. Severe hemophiliacs, who number about 10,000 in the United States, can be treated with infusion of human factor VIII, which will restore the blood's normal clotting ability if administered with sufficient frequency and concentration. The classic definition of factor VIII, in fact, is the substance present in normal blood plasma that corrects the clotting defect in plasma derived from individuals with hemophilia A.
Several preparations of human plasma-derived factor VIII of varying degrees of purity are available commercially for the treatment of hemophilia A. These include a partially purified factor VIII derived from the pooled blood of many donors that is heat- and detergent treated for viruses but contain a significant level of antigenic variants; a monoclonal antibody purified factor VIII that has lower levels of antigenic impurities and viral contamination; and recombinant human factor VIII.
The development of antibodies (“inhibitors” or “inhibitory antibodies”) that inhibit the activity of factor VIII is a serious complication in the management of patients with hemophilia.
Alloantibodies develop in approximately 20% of patients with hemophilia A in response to therapeutic infusions of factor VIII. In previously untreated patients with hemophilia A who develop inhibitors, the inhibitor usually develops within one year of treatment. Additionally, antibodies (autoantibodies) that inactivate factor VIII occasionally develop in individuals with previously normal factor VIII levels. If the inhibitor titer is low enough, patients can be managed by increasing the dose of factor VIII. However, often the inhibitor titer is so high that it cannot be overwhelmed by factor VIII. An alternative strategy is to bypass the need for factor VIII during normal hemostasis using activated prothrombin complex concentrate preparations (for example, KONYNE (Cutter Laboratories), FEIBA (Baxter Healthcare), PROPLEX (Baxter Healthcare)) or recombinant human factor Vila. Additionally, since porcine factor VIII usually has substantially less reactivity with inhibitors than human factor VIII, a partially purified porcine factor VIII preparation (HYATE: C (IPSEN Pharma)) has been used. Many patients who have developed inhibitory antibodies to human factor VIII have been successfully treated with porcine factor VIII and have tolerated such treatment for long periods of time.
However, public health concerns regarding the risk of viruses or other blood-borne contaminants have limited the usefulness of porcine factor VIII purified from porcine blood. A recombinant porcine factor VIII variant has therefore been developed, which is designated “OBI-1” and described e.g. in WO 01/68109. OBI-1 is a partially B-domain deleted porcine FVIII. This molecule is presently in clinical development.
B-domain deleted factor VIII variants are known to keep the procoagulant and cofactor activity of factor VIII. In addition to this, Mertens et al. (British Journal of Haematology 1993, 85, 133-142) describe recombinant human factor VIII variants lacking the B-domain and the heavy-chain sequence spanning from Lysine 713 to Arginine 740.
Many hemophiliacs require daily replacement of factor VIII to prevent bleeding and the resulting deforming hemophilic arthropathy. In view of this, there is a need for a more potent factor VIII molecule.